DESCRIPTION: The overall hypothesis is that mu opioid receptor (MOR) endocytosis and recycling helps determine how the guinea pig ileum enteric neurons respond to endogenous opioids and alkaloids. The specific aims are (1) to localize MOR in functional types of myenteric neurons, describe the anatomical relationship between MOR and enkephalins and determine whether endogenous release of enkephalins can produce MOR endocytosis, (2) delineate the intracellular pathway of ligand-induced MOR endocytosis and determine if it returns to the surface, (3) determine whether there is a temporal and dose correlation between MOR endocytosis and the opioid induced reduction in excitatory enteric neurotransmission as measured by the diminution of the muscle twitch, release of ACh and SP and SP internalization using LMMP preparation, (4) to test the hypothesis that MOR internalization alters the effect of opioids on enteric neurons by using inhibitors of receptor endocytosis/recycling and pertussis toxin to inactivate inhibitory G protein pathways and measuring the diminution of the muscle twitch, release of ACh and SP and SP internalization. The project will use a variety of in vivo and in vitro preparations, with an emphasis on light electron and confocal microscopy. It will show the location of mu opioid receptor (MOR) immunocytochemically in excitatory and inhibitory neurons, and determine the cellular pathways by which MOR endocytosis and recycling occurs using a variety of histological cellular markers and pharmacological tools. The functional significance of this will be investigated by correlating the MOR endocytosis with opioid-induced inhibition of excitatory neurons and the responsiveness of longitudinal muscle-myenteric plexus preparations. These studies will provide information on how MOR receptor plays a role in peristaltic control and the mechanisms of cellular processing of MOR in response to opioid ligands. Information on sequestration and cellular handling of the MOR will provide insight into mechanisms for receptor desensitization and eventual down-regulation.